This invention is in the general area of genetic engineering of nucleic acid sequences, especially RNA sequences that are substrates for Ribonuclease P.
There are several classes of ribozymes now known which are involved in the cleavage and/or ligation of RNA chains. A ribozyme is defined as an enzyme which is made of RNA, most of which work on RNA substrates. Ribozymes have been known since 1982, when Kruger, Kelly, et al., "Self-Splicing RNA: Autoexcision and Autocyclization of the Ribosomal RNA Intervening Sequence of Tetrahymena, " Cell 31:147-157 (1982) showed that a ribosomal RNA precursor in Tetrahymena, a unicellular eukaryote, undergoes cleavage catalyzed by elements in the RNA sequence to be removed during the conversion of the rRNA precursor into mature rRNA. Another class of ribozyme, discovered in 1983, was the first to be shown to work in trans (i.e., to work under conditions where the ribozyme is built into one RNA chain while the substrate to be cleaved is a second, separate RNA chain). This ribozyme, called M1 RNA, was characterized in 1983 by Altman and colleagues as responsible for the cleavage which forms mature 5' ends of all transfer RNAs (tRNAs) in E. coli. Analogous RNA-containing enzymes concerned with tRNA synthesis have since been found in all cells in which they have been sought, including a number of human cell lines, though the relevant eucaryotic RNAs have not yet been shown to be catalytic by themselves in vitro.
The discovery and characterization of this catalytic RNA is reviewed by Sidney Altman, in "Ribonuclease P: An Enzyme with a Catalytic RNA Subunit" in Adv. Enzymol. 62, 1-36 (1989). The activity was first isolated from E. coli extracts, and subsequently determined to be a ribonucleoprotein having two components, an RNA component called M1 and a protein component called C5. The RNA cleaved substrates in a true enzymatic reaction, as measured using Michaelis-Menton kinetics. M1 was determined to be solely responsible for substrate recognition and C5 was determined to alter k.sub.cat but not K.sub.M, as reported by Guerrier-Takada, et al., Cell 35, 849 (1983) and McClain, et al., Science 238, 527 (1987). Sequencing showed that M1 RNA is 377 nucleotides long, M.sub.r approximately 125,000, and that the protein consists of 119 amino acids, M.sub.r approximately 13,800, as reported by Hansen, et al., Gene 38, 535 (1987).
Cleavage of precursor tRNA molecules by the RNA component of eubacterial RNAase P is described by Guerrier-Takada, et al., Cell 35, 849 (1983) and reviewed by Altman, Adv. Enzymol. 62, 1 (1989).
U.S. Pat. No. 5,168,053 entitled "Cleavage of Targeted RNA by RNAAse P" to Altman, et al., discloses that it is possible to target any RNA molecule for cleavage by bacterial RNAase P by forming a nucleotide sequence part of which is complementary to a targeted site and which includes a terminal 3'-NCCA, wherein the sequence is designed to hybridize to the targeted RNA so that the bacterial RNAase P cleaves the substrate at the hybrid base-paired region. Specificity is determined by the complementary sequence. The sequence is preferably ten to fifteen nucleotides in length and may contain non-complementary nucleotides to the extent this does not interfere with formation of several base pairs by the complementary sequence which is followed by NCCA at the 3' end.
Subsequent studies have demonstrated that the external guide sequence, or "EGS", that is useful in targeting procaryotic RNAase P, does not result in cleavage of a targeted RNA strand by eukaryotic RNAase P.
It is therefore an object of the present invention to provide methods and compositions for specifically cleaving targeted RNA sequences using eukaryotic RNAase P or functional equivalents thereof.
It is a further object of the present invention to provide methods and compositions for specifically cleaving RNA, both in vitro and in vivo within eukaryotic cells, for the treatment of disease conditions which involve RNA transcription or translation, such as diseases caused by RNA and DNA viruses and expression of excessive or pathogenic proteins from mRNA, or of excessive or pathogenic RNA, itself.